Abnormalities of stem cell function and/or the destruction of stem cells leads to an olfactory epithelium (OE) that remains olfactory (including a full complement of sustentacular cells), but one that has no neurons, no globose basal cells, and no ongoing neurogenesis. Horizontal basal cells (HBCs) are still evident in aneuronal OE, but clearly are not functioning as multipotent progenitors/stem cells as they can and do under other circumstances. These and other data indicate that HBCs must be activated in some way by signals emanating from a damaged epithelium, in order to function as stem cells. Recently, we have shown that the transcription factor p63 is key to HBC differentiation and regeneration. We will test whether p63 is the key factor that regulates (prevents) HBC activation using a mouse genetic approach. In Aim 1 we will conditionally knock-out and conditionally over-express p63 selectively in HBCs, using K5 driver lines, and analyze clonal progeny from the targeted HBCs. Signaling via the Wnt and Notch pathways play antagonistic roles in regulating stem cells and their transition to more differentiated and shorter-lived progenitors. Moreover, both of them interface with p63 in that regulation. Accordingly, in Aims 2 and 3, we will assess how knock-out vs. constitutive activation of the Notch and Wnt pathways, respectively, affect HBC activation and function as progenitor. We will use the same kind of approach to achieve cell type- and temporal-control over the targeted genes. We expect that the results will show that p63 is the master regulator of HBC activation and that both Wnt and Notch pathways will participate in controlling both p63 and HBC activation. After completion of the experiments, we will be poised to apply small molecule inhibitors of p63 and the key signaling pathways to the problem of restoring neurogenesis to an aneuronal OE. PUBLIC HEALTH RELEVANCE: Olfactory dysfunction is a nearly universal consequence of aging and has serious consequences on nutritional status as well as quality of life more generally. In some cases, exhaustion and/or inappropriately prolonged dormancy of the stem cells of the olfactory epithelium looks to be the culprit. The experiments proposed in this grant test how olfactory stem cells can be awakened in mice so that the epithelium is restored to health. We will manipulate a key transcription factor, p63, that acts as the master switch for one category of olfactory stem cells, as well as the Notch and Wnt pathways that act on p63. When completed, we can use the information gained to design potential therapeutic approaches to reinvigorate stem cells.